F. OWEN and RACHELC. BOURN% Division of Psychiatry, Clinical Research Centre, Harrow HA1 WJ, England

and J. C. K. LAI and R. WILLIAMS Liver Research Unit, Kings College Hospital, London, England

Abstract-Three

~o~uiations of rat brain rn~tocho~d~a were isolated by ccntr~fu~at~on in a Ficollsucrose discontinuous gradient. The three fractions showed differential activity towards ~-h~dro~~try~iamine (a type A MAO substmte) and phenyl~t~ylam~n~ {a type B MAO substrate). There were also clear differences in the inhibition characteristics of MAO with the specifiic inhibitors clorgyline and deprerryl, It is suggested that rat brain mitochondria are not homogeneous with respect to their complements of MAO. and that the differences are not due to artefacts produced by the isolation procedure.

Different preparations of brain mitochondria may be enriched with some enzymes and deficiennt in others tl, 2,3,4,5]. In addition, it is possible to distinguish betwveenmifochondria derived from neuronal and gfiai cells [6] and synaptic and non-synaptic mitochondria [I, 43. The evidence for the heterogeneity of brain mitochondria has been reviewed [Q. Monoamine oxidase (MAO, monoamine-O, oxidoredu~t~s~, EC 1.4.3.4) is bound to the outer mitochondr~al membrane Eg]. There is considerably evidence that it exists in more than one form: several bands containing MAO activity have been resolved on poiyacrylamide gel eiectrophoresis of solubilised preparations from rat brain mitochondria 1193.On the basis of in virro studies with the MAO inhibitor clorgyline (IV-methyl-N-propargyl-3-(2,4-dichlorophenoxypropylamine)), Johnstone [la] suggested two forms of MAO, type A and type B, in rat brain. In virrt, type A MAO is relatively sensitive to inhibition by clorgyline [$O] and type B MAO to deprenyl (phenylisopropylmethylprop~ny~~~ne~ ti If. Noradrenaiine [X2] and 5-hydroxytryptam~ne IlO] are preferentially d~~jnat~d by type A MAO, and benzylamine Et31 and ~-pheny~ethy~ami~e [14] by type B MAO. Tyramine [IO] is a substrate for both forms of the enzyme. Tipton and Houslay [15]. however, have suggested that the substrate preferences, inhibition characteristics and multipfe forms of MAO are attributable to the adherence to the enzyme of differing quantities of Iipid or membraneous material. Lai et ai_ [ 161 reported a technique for the preparation of three distinct populations of rat brain mitochondria that were reiativety free from non-mitochondriat contamination. Two of these populations were derived from synaptosomes, and the remainder were free mitochondria. The mitochondrial populations appear to differ from each other in certain aspects uf metabolism of citric acid cycle and related intermediates. In view of this heterogeneity, we investigated 289

the characteristics of monoamine oxidase in the three mitochondrial populations, MATERIALS AND METHODS Chemicibls. Tyramine, S-hydr~xytrypt~mi~e creatinine sulphate, ~-ph~ny~~thy~am~~e and mannitol were obtained from Sigma (London). ~~4C~tyram~n~ hydrochloride and [~4CJ5-hydro~ytryptamin~ creatinine sulphate were obtained from the Radio~hem~~a~ Centre, Amersham, England, and ~~4C~~-pheny~~thy~amine hydrochloride from ICN Pharmaceutical Inc., California, U.S.A. C.lorgyline was obtained from May and Baker Ltd., Dagenham, Essex and Deprenyl was a kind gift from Dr. A. R. Green, Oxford University. Al1 other chemicals were from British Drug Houses Ltd., Poole, England and of the highest purity available. FicoIl was dialysed against glass-distilled water for at least 5 hr before use. Prepuzztion of ~ito~~~~dr~~~~ popufariorzs. The method employed to prepare the m~t~hondrial populations was essentially similar to that described by Lai [I73 and Lai et ai. [la), except that the final wash of the three mi~o~hond~ia~ fractions with bovine plasma albumin medium was omitted. Male Wistar rats Cl&200 g) were guillotined, the brains removed and transected at the level of the collicuii. That part of the brain rostra1 to the transection (ie. the forebrain), with the exception of the aifactory bulbs, was fineiy minced with scissors. Excess blood was removed by washing the minced brain in isoIafion medium (0.32M sucrose, 1 mM potassium-EDTA and 10 mM Tris-HCl pH 7.4) and decanting off the supernatant. A IO:& g J:

in isolation

4

medium

supernatant

min supernatant

combined supernatant

Pellet*

w 4 17,000 g x IO min I

crude mitochondrial pellet (CM) resuspended in 15 ml solation medium t FicallS”CPOSe media

I supernatant

Synaptosomal

fraction

(Syn) diluted

I

with 3 ~01s isolation

medium

+ 18,500 g x IO mln

I

pellet resuspended ml isolation medium

Pellet

lysed

with 30 ml pH 8.1 at 2’C

mitochondria

50,000 g x 40 min

18,500 g x 10 min + Pellet resuspended 10 ml 6m~ Tris-HCl pH 8.1

with

3,300 g x 10 min

Pellet resuspended with4 10 ml 3% Ficoll-Sucrose Medium

S

diluted

11,

B,OO g

with equal medium

21 .. isolation x

10 min

t Pellet resuspended isolation medium

Pellet

below G?; Flcoll-Sucrose

(P)

Synaptic mitochondria

Pellet (PI &suspended in 3 ml isolation medium + Synaptic

mitochondria

Fig. 1. Preparation

SM

SM2

of synaptic

and non-synaptic

rat brain

mitochondria

in 3 ml

291

Monoamine oxidase in distinct populations of rat brain mitochondria

Table 1. MAO activity of mitochondrial fractions using 5-hydroxytryptamin~ and phenylethylamine as substrates S-fIydroxytryptamine

Fraction

s

Phenytethylamine

15.6 + 7.5

51.6 i 16.4 117.5 + 16.6

8 8

31.7 + 5.8 51.8 It 4.6

106.6 + 3.0

SM2

(MAO activity, mean + S.D., expressed as nmoles product/mg

protein,‘30

min).

organic phase was added to 1Oml of Triton Xl00 based scintilIant and the deamina~ed products quantified by scintillation counting. Blanks consisted of boiled mitochondrial preparations carried through the entire procedure. Mitochondrial protein was measured using a phenol reagent technique [20) and MAO activity was expressed as nmoles of product formed/mg protein/30 min. ~~~~b~~~o~ stwdies. MAO activity was determined as described with [‘4CJtyramine as substrate with increasingcon~entrationsof~iorgyline( 10-14M-10i “M) and deprenyl (10-~~-l~-3M). Prior to the MAO assay incubation mixtures were preincubated at 37” for IOmin in the presence of inhibitor but in the absence of substrate.

RESULTS MAO u&city. The specific activities of MAO, with 5-hydrox~tryptamine and phenylethyiamine as substrates, in the three mitochondrial fractions are ptesented in Table 1. From Table 1 it is clear that the three mitochondrial fractions were heterogeneous with respect to MAO activity and fell into three distinct groups when phenylethylamine was used as substrate. Both synaptosomal fractions (SM and SM2) more actively deaminated 5-hydroxytryptamine than did the free mitochondria (M). 1}7kibiti(~tlstudies. The effects of increasing concentrations of cforgyline and deprenyl on the MAO acti-

14

12

Concentration

10 of ciorgyline

8

6

4

(-log concentration

2 M)

Fig. 2. Effect of clorgyline concentration on MAO activity of rat brain mitochondriaf fractions with tyamine as substrate. (CJ--CM-crude mitochondda, I-SM-light synag tosomal mitochondria, @-SMZ-heavy synaptosomal mitochondria, +--M-free mitochond~a).

I IO

9

8

7

6

5

4

3

Concentration of deprenyi Hog concentration M)

Fig. 3. Effect of deprenyl concentration on MAO activity of rat brain mitochondrial fractions with tyramine as substrate. (D-CM-crude mitochondria, C-SM-light synaptosomal mitochondria, @-SMZ-heavy synaptosomal mitochondria, +--M-free mitochondria).

vity of the m~to~hondrial fractions are presented in Figs. 2 and 3 respectively. A double sigmoid curve. indicative of two forms of MAO, resulted in all fractions, with the SM fraction being particularly sensitive to inhibition by clorgyline. The differences in inhibition characteristics between the mitochondrial fractions were not as marked with deprenyl as with clorgyline. DISCUSSION

The three populations of rat brain mi~ochondria described by Lai et al. [i6] differed markedly from each other with respect to monoamine oxidase activity when both the A, and B forms of the enzyme were taken into account. It is noteworthy that all the mitochondria1 fractions showed considerably more type A activity than type B, particularly the synaptosomal fractions. Serotonin and noradrenaline are preferentialiy deaminated by type A MAO and recent evidence presented by Braestrup, Anderson and Randrup [2if suggests that this may also be the case for dop~ine. Differential type A and type B activities have been reported in mitochondrial fractions after centrifugation in sucrose gradients 122,231. The results of the inhibition studies with clorgyiine and deprenyl strongly supported the finding of differential type A and type If MAO activities in the mitochondrial popdations. The clorgyline inhibition curves showed distinct differences in percentage inhibition at their plateaus, indicating relatively different activities of type A and type B monoamine oxidase. The deprenyt inhibition curves did not show such striking differences between the three fractions but did support the vahdity of the clorgyline inhibition curves in as much as that the SM fraction, which was most sensitive to inhibition by clorgyline, was the least sensitive to inhibition by deprenyl and vice versa for the SM2 fraction. It appears, therefore. that not only is monoamine oxidase heterogeneous in preparations of crude mitochondria from rat brain but that the mito~~ondria themselves are not all similar with respect to their

292

F. OWEN, R. C. BOURNE,J.

MAO content. However, as Wousfay and Tipton [IS] have pointed out these differences need not be the result of different enzyme proteins and may weil be due to the presence of different quan~jti~s or classes of fipids or type of birding of the enzyme to the mitochondrial membrane. Since intact mito~hondria were used in the present study. it seems unlikely that the results obtained were due to artefacts produced by preparation of the fractions. and differential MAO activity towards a variety of substrates is likely to be an in &o reality. The ~i~~r~utia1 in ciro metabolism of brain amines after administrat~o~l of specific MAO inhibitors has been demonstrated by Neff et LIL.[22]. Whether or not the two synaptosomal mitochondrial populations are representative of mitochondria from different anatomical regions of the rat brain is at present unknown, although the technique used in the present i~~vestigation could be scaled down and made suitable for investjgatjng this possibility. A~kaow,ledyrmP,tts--We are grateful to the Rothschild Schizophrenia Research Fund for defraying the salary of F.O.

C. K.

5. M. C. Kroon, PhD Thesis. University of Leiden (1972). 6. A. Hamberger, C. Blomstrand and ‘4. L. Lehninger, J. Cell Bioi. 45, 221 (1970). 7. C. J. Van den Berg, in ,~f~iub~~lj~Corrtparrmer~fafir~n in the Brain (Eds. R. Balazs and J. E. Cremer.) pp. 137-166. Macmillan, London (1973). 8. C. Schnaitman, V. G. Erwin and J. W. Greenawalt, J. Ceil Biol. 32, 719 (1967).

M. B. H. Youdim. C. G, S. Coltins and M. Sandier. .“iaturr 223, 626 (1969). 10. J. P. Johnston. &o&mm. Pharmau. 17, 1285 (1968). i 1. 1. Knoll and I(. Magyar. pi& ~iorhem. Fs~e~~op~urmuc. 5, 393 (1972). 12. C. Gcridis and N. H. Neff, Br. f. Pharmac. 43, 814 9.

(1971). 13. 14.

16.

17. 18.

20. 12,

287 (1565). 2. C. M. .l. Van Kempen, C. .I. Van den Berg, H. J. Van der Helm and H. Veldstra, jr. ~~1~~~~~~~2. 12, 581 (1965). 3. R. Balazs, D. Dahl, and J. R. Harwood, J. ~e~r~ichet~. 13, 897 (1966). 4. G. G. D. Blokhuis and H. Veldstra., FEBS Lrfr. 11, 197 (1970).

A. J. Christmas, C. J. C&son, D. R. Maxwell and D. Riddell, Br. J. Pharntac. 45, 490 (1972). H. Y. T. Yang and N. H. Neff, J. Phurmuc. exp. Ther.

187, 365 (1973). 15. M. D. Houslay

19.

I. L. Salpanicoff and E. De Robertis. J. Nrurochtm

LAI and R. WU.I.IAMS

21.

and K. F. Tipton, &o&m. J. $35, 173 iI973). J. C. K. Lai, 3. M. Walsh, S. C. Dennis and J. B. Clark, in Metabolic Cl~nlpa~~rnenfa~~~~~ and Neurotransmission (Eds. S. Berl and D. Schneider) pp. 487-496. Plenum, New York (1975). J. C. K. Lai. PhD Thesis, University of London (1975). J. C. K. tai, J, NI. Walsh. S. C. Dennis and J. B. Clark, J. .~~,~~ochettt. in press. D. S. Robinson, W. Lovenberg, H. Keiser and A. Sjoerdsma. Biochem. P~ta~muc. 17, 109 (1968). E. W. Sutherland, C. F. Cori, R. Haynes and N. S. Olsen. J. hiol. Chrm., 180, 825 (1949). C. Braestrup, H. Anderson and A. Randrup, Eur. J.

Pharmuc. 34, 18I 22. N. H. Neff, H. Y. ps~fhopiurrmurol~

(I 975).

T. Yang and J. A. Frentes, in Neuroqf‘ ~o~~f~~i~l~s uad their Rrguiatory Enz~tnmes(Ed. E. Usdin), pp, 49-50. Raven Press, New York (1974). 23. M. C. Kroon and H. Veldstra. FEBS Lett. 24. 173 (1972).